The aim of this project is to elucidate the physiological and pharmacological properties of mammalian presynaptic nerve terminals. Pinched-off terminals (synaptosomes) from rat brain are used for these studies. In one type of study, tracer 45Ca flux methods and fluorometric methods (with a Ca-sensitive fluorochromic indicator) will be employed to determine how the smooth endoplasmic reticulum (SER), mitochondria and the plasma membrane interact to help regulate the cytoplasmic free Ca2+ concentration, and to determine how these organelles respond to the changes in free Ca2+ that occur with neuronal activity. The resulting data should enable us to model these interrelationships accurately. In the second type of study, which comprises the major portion of the proposed program, the physiological, pharmacological and biochemical properties of the various potassium channels that are present in nerve terminals will be characterized. Tracer (86Rb) efflux studies will be used to determine the sensitivities of the K channels to various drugs and toxins; the channels will be incorporated into planar lipid bilayers to study the kinetic and pharmacologic properties of single K channels; the binding characteristics of K channel blockers and modulators will be investigated in synaptic membranes; the pharmacological properties will then be used to help identify the K channel proteins that will be isolated, characterized, and reconstituted into lipid bilayers to verify their functional integrity. The first K channel that will be investigated in this way is the non-inactivating, voltage-regulated K channel. This channel has been identified as the high-affinity phencyclidine (PCP) receptor (about 95,000 and 80,000 Dalton MW) in the brain. The results may help to explain how PCP induces a schizophrenia-like syndrome. We will then use similar methods to investigate the properties of a K channel that is activated by certain opiates.